Colony-forming Units In Culture Research Articles

Evaluating models and assessment techniques for understanding oral biofilm complexity.

Oral biofilms are three-dimensional (3D) complex entities initiating dental diseases and have been evaluated extensively in the scientific literature using several biofilm models and assessment techniques. The list of biofilm models and assessment techniques may overwhelm a novice biofilm researcher. This narrative review aims to summarize the existing literature on biofilm models and assessment techniques, providing additional information on selecting an appropriate model and corresponding assessment techniques, which may be useful as a guide to the beginner biofilm investigator and as a refresher to experienced researchers. The review addresses previously established 2D models, outlining their advantages and limitations based on the growth environment, availability of nutrients, and the number of bacterial species, while also exploring novel 3D biofilm models. The growth of biofilms on clinically relevant 3D models, particularly melt electrowritten fibrous scaffolds, is discussed with a specific focus that has not been previously reported. Relevant studies on validated oral microcosm models that have recently gaining prominence are summarized. The review analyses the advantages and limitations of biofilm assessment methods, including colony forming unit culture, crystal violet, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt assays, confocal microscopy, fluorescence in situ hybridization, scanning electron microscopy, quantitative polymerase chain reaction, and next-generation sequencing. The use of more complex models with advanced assessment methodologies, subject to the availability of equipment/facilities, may help in developing clinically relevant biofilms and answering appropriate research questions.

Aloe vera mouthwashes can be a natural alternative to chemically formulated ones – A randomized-controlled trial

ObjectivesTo evaluate the antiplaque and antibacterial efficacy of commercially available mouthwashes containing aloe vera (AV), hydrogen peroxide (HP), and cetylpyridinium chloride (CPC) in a 4-day plaque regrowth study. MethodsPlaque score and salivary samples were assessed (Day-0 and Day-4) in 96 participants in a randomised, double-blind prospective parallel-arm 4-day plaque regrowth study. Participants were divided into five groups who refrained from engaging in regular oral hygiene measures during the study period and used commercially available mouthwashes containing AV, HP, and CPC as test products with distilled water (DW) and chlorhexidine (CHX) mouthwash as negative and positive controls, respectively. Salivary bacterial count was expressed as colony-forming units (CFU) (culture method). ResultsThere was a significant difference both in plaque score (p < 0.001) and in CFU (p < 0.001) among the study mouthwashes at Day-4. The plaque score and CFU of AV were significantly higher and lower than those of CHX and DW, respectively. The plaque score of HP was significantly higher than that of AV (p = 0.016) and CPC (p < 0.001). No significant difference was observed between AV and CPC (p = 0.70). Moreover, the CFU of HP was significantly higher than that of CPC (p = 0.04). There was no statistically significant difference between the CFU of mouthwashes containing AV and HP (p = 0.912) or AV and CPC (p = 0.280). No significant difference was seen in the inhibition of plaque and salivary bacterial count between AV, HP, and CPC. ConclusionThe antiplaque and antibacterial efficacy of commercially available AV mouthwash was similar to that of CPC and significantly better than that of HP mouthwash and can be a natural alternative to chemically formulated mouthwashes.

Efficacy of the dual-action GA-KR12 peptide for remineralising initial enamel caries: an in vitro study.

To investigate the antibiofilm and remineralising effects of the dual-action peptide GA-KR12 on artificial enamel caries. Enamel blocks with artificial caries were treated with sterilised deionised water as control or GA-KR12. The blocks underwent biochemical cycling with Streptococcus mutans for 3weeks. The architecture, viability, and growth kinetics of the biofilm were determined, respectively, by scanning electron microscopy (SEM), confocal laser scanning microscopy, and quantitative (culture colony-forming units, CFUs). The mineral loss, calcium-to-phosphorus ratio, surface morphology, and crystal characteristics of the enamel surface were determined, respectively, using micro-computed tomography, energy dispersive spectroscopy, SEM, and X-ray diffraction (XRD). SEM showed confluent growth of S. mutans in the control group but not in the GA-KR12-treated group. The dead-to-live ratios of the control and GA-KR12-treated groups were 0.42 ± 0.05 and 0.81 ± 0.08, respectively (p < 0.001). The log CFUs of the control and GA-KR12-treated groups were 8.15 ± 0.32 and 6.70 ± 0.49, respectively (p < 0.001). The mineral losses of the control and GA-KR12-treated groups were 1.39 ± 0.09gcm-3 and 1.19 ± 0.05gcm-3, respectively (p < 0.001). The calcium-to-phosphorus molar ratios of the control and GA-KR12-treated groups were 1.47 ± 0.03 and 1.57 ± 0.02, respectively (p < 0.001). A uniformly remineralised prismatic pattern on enamel blocks was observed in the GA-KR12-treated but not in the control group. The hydroxyapatite in the GA-KR12-treated group was better crystallised than that in the control group. The dual-action peptide GA-KR12 inhibited the growth of S. mutans biofilm and promoted the remineralisation of enamel caries. GA-KR12 potentially is applicable for managing enamel caries.

Signal-Transducing Adaptor Protein-2 Blocks B Cell Recovery Under Hematological Stress at Pre-B Stage Via TLR4 Signaling

All lineages of blood cell production from hematopoietic stem cells (HSC) are maintained for an entire lifetime. That is consistent under homeostatic condition, while hematopoiesis is changed in response to various types of stress, including infections and aging. It has been reported that hematopoietic stem and progenitor cells (HSPC) are targets of pathogen products and danger signals. After the exposure to Gram-negative lipopolysaccharide (LPS) that is the ligand of Toll-like receptor (TLR) 4, HSC enter cell-cycle and differentiate into myeloid lineage cells while B lymphopoiesis is suppressed. Although the regulation of hematopoiesis by HSC is well studied, many questions still remain regarding how progenitors contribute the response to hematopoietic demands. Signal-transducing adaptor protein-2 (STAP-2) was cloned as a c-fms/M-CSFR interacting protein in 2003, and the interaction with a variety of signaling or transcriptional molecules such as STAT5, MyD88 and IκB kinase (IKK) in macrophages or T cells has been reported. The function as an adaptor protein in various cell types and signaling pathways, and its ubiquitous expression promoted us to investigate the effect of STAP-2 on HSPC under hematopoietic stress using gene-modified mice.We previously reported that hematopoiesis in STAP-2 knock out mice (KO) is normal under homeostatic condition. First in this study, competitive bone marrow (BM) transplantation experiment was conducted using KO. Four months after intravenous injection of Lineage- Sca1+ cKithigh (LSK) HSPC (CD45.2+) derived from KO or wild-type mice (WT) with competitor BM mononuclear cells (CD45.1+), the engraftment and lineage distribution were comparable, indicating that STAP-2 does not affect the function of long-term HSC. Interestingly, we found that chimerism in peripheral B cells at 1 month after transplantation in KO LSK transplanted mice was higher than control with statistical significance. In BM, STAP-2 deficiency increased pre-B and immature B cell cells, while there were no differences in numbers of LSK HSPC or Lin- Sca1+ cKitlow Flk2high common lymphoid progenitor (CLP) between WT and KO donor. To evaluate the role of STAP-2 in early B lymphopoiesis, we generated transgenic mice (Tg) that overexpress STAP-2 under the control of Eμ enhancer and Lck proximal promoter. The promoter could drive expression of the inserted cDNA in lymphoid lineage cells from CLP stage. In Tg, we observed significant decrease of B progenitors compared to WT BM. When HSPC (LSK or CLP) derived from Tg were cultured with OP9 stromal cells under B cell condition in the presence of IL-7, Flt3-ligand and SCF, the production of B220+ B cells were significantly suppressed. The same was true in stromal-cell free cultures. In colony-forming unit (CFU) assays, we found that STAP-2 overexpression decreased the ability to generate pre-B colonies, while the deficiency promoted high recovery with statistical significance (recovered colony number; 40.0 ± 1.4 in WT, 7.0 ± 1.4 in Tg, and 89.0 ± 5.7 in KO). To elucidate the detailed mechanisms, RNA-seq experiment with WT and Tg under steady-state as well as WT and KO 1 month after transplantation was conducted. Data among them were compared, and bioinformative approach with Ingenuity Pathway Analysis revealed that STAP-2 upregulates TLR4 signaling in pre-B cells during stress hematopoiesis. Consistent with the results, when LPS was added into CFU cultures, the recovered pre-B colony was suppressed. Moreover, deletion of STAP-2 partially cancelled the inhibitory effect of LPS on pre-B cell proliferation.In summary, we found that STAP-2 regulates the early B lymphopoiesis under hematopoietic stress. While STAP-2 did not influence stemness of HSC or homeostatic hematopoiesis, short-term recovery of B cells after transplantation was promoted by STAP-2 deficiency. This effect was induced by the activated proliferation of pre-B cells. Analyses with Tg mice confirmed the inhibitory effect of STAP-2 on early B lymphopoiesis. RNA-seq analyses have shown that activation of TLR4 signaling at pre-B stage occurs 1 month after transplantation as well as in Tg mice, and STAP-2 deficiency partially cancelled the inhibitory effects of LPS in CFU pre-B assays. Our study unveiled the critical role of lineage progenitors in short-term response to hematological demands and developmental stage-specific regulation of TLR4-STAP-2 pathway. DisclosuresOritani:Celgene: Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau; MOCHIDA PHARMACEUTICAL: Speakers Bureau; Novartis Pharma: Consultancy, Speakers Bureau. Shibayama:Takeda Pharmaceutical Co.,LTD.: Honoraria, Research Funding; Fujimoto Pharmaceutical Co.: Honoraria, Research Funding; Jansen Pharmaceutical K.K.: Honoraria; Bristol-Meyer Squibb K.K.: Honoraria, Research Funding; Ono Pharmaceutical Co.,LTD.: Honoraria, Research Funding; Novartis Pharma K.K.: Honoraria, Research Funding; Mundipharma K.K.: Honoraria, Research Funding; Celgene K.K.: Honoraria, Research Funding. Kanakura:Alexion Pharmaceuticals, Inc.: Honoraria, Research Funding; Bristol Myers: Research Funding; Fujimotoseiyaku: Research Funding; Chugai Pharmaceutical: Research Funding; Astellas: Research Funding; Pfizer: Research Funding; Nippon Shinyaku: Research Funding; Toyama Chemical: Research Funding; Shionogi: Research Funding; Eisai: Research Funding; Kyowa Hakko Kirin: Research Funding.

Влияние лиофилизации на сохранность структурнофункциональных характеристик лейкоконцентрата кордовой крови человека

This paper presents the investigations on the impact of lyophilisation conditions on structural and functional characteristics of human cord blood leukoconcentrate (HCBL) such as: the number and viability, cytomorphological indices, content of hematopoietic stem (CD34+) and T-regulatory cells (CD4+CD25high), hematopoietic colony forming units in culture (CFUc). Four regimens, including two programs with and without 3.5% DMSO were used for hcbl cell lyophilisation. The programs dii¬€ered in temperature value and duration wherein the primary drying proceeded. When using the i¬rst program, the cells were dried for 10 hrs (–28ÂoÐi), and 9 hrs (–18ÂoÐi) for the second one. The designed method for HCBL lyophilisation, suggested the use of the DMSO-free i¬rst program, ensured the cell preservation in a heterogeneous population of cord blood according to all the studied characteristics. Probl Cryobiol Cryomed 2019; 29(4): 332-343

Modeling and Simulation of Pretomanid Pharmacodynamics in Pulmonary Tuberculosis Patients.

Pretomanid (PA-824) is a nitroimidazole in clinical testing for the treatment of tuberculosis. A population pharmacodynamic model for pretomanid was developed using a Bayesian analysis of efficacy data from two early bactericidal activity (EBA) studies, PA-824-CL-007 and PA-824-CL-010, conducted in Cape Town, South Africa. The two studies included 122 adult male and female participants with newly diagnosed pulmonary tuberculosis who received once daily oral pretomanid doses of either 50, 100, 150, 200, 600, 1,000, or 1,200 mg for 14 days. The structural model described capacity-limited growth and saturable drug-induced bacterial killing with separate rate equations for sputum solid culture colony forming unit (CFU) counts and liquid culture time to positivity (TTP) that were linked through a time constant. The posterior population geometric means and interindividual variability percent coefficients of variation were, respectively; 0.152±0.013 log10 CFU/mL sputum/day and 54%±6% for the maximum kill rate constant, 20.4±1.0 h and 20.8%±0.1% for the time constant of proportionality between the CFU and TTP rate equations, and 770±140 ng/mL and 48%±17% for the pretomanid half-maximum effect plasma concentration. Model simulations showed once daily pretomanid at 100 mg, 200 mg, and 300 mg, attained 58%, 73%, and 80%, respectively, of an expected maximum 14-day EBA of 0.136 log10CFU/mL sputum/day. These results establish a pretomanid exposure-efficacy relationship with dual outcomes for CFU counts and TTP, and with potential applications to dose optimization of pretomanid-containing regimens.

GPR56 AND GPR97 PLAY REDUNDANT ROLES IN REGULATING HEMATOPOIESIS

Hematopoietic stem cells (HSCs) are responsible for the lifelong production and maintenance of adult blood cell types. They arise from the aorta at the embryonic day (E) 10.5 from a subset of endothelial cells that undergo to endothelial-to-hematopoietic transition (EHT). Our lab discovered that a G-coupled receptor protein, Gpr56, is required for the generation of HSCs in zebrafish. However, its role in mammalian hematopoiesis remains controversial. We investigate the functional role of Gpr56 in vitro using our mouse Gata2Venus (G2V) embryonic stem cell (ESC) line which allows us to highly enrich for hematopoietic progenitor cells (HPCs). G2V.G56-/-. We tested the Gpr56 loss of function by generating a full knock out (KO) Gpr56 mouse G2V (G2V.G56-/-) ESC line. The in vitro examination of phenotypic hematopoietic cell production by FACS and colony forming unit-culture (CFU-C) assay reported an increase in hematopoietic cells (HC) and HPCs production from G2V.G56-/- ESC as compared to G2V ESC. Further analyses performed on cell-sorted HPC population showed that another GPR protein, Gpr97 (located in the same locus), is highly expressed when Gpr56 is deleted. This led us to generate a novel G2V Gpr56/Gpr97 double knockout (G2V.G56/97-/-) ESC line. The in vitro differentiation of the G2V.G56/97-/- revealed a significant decrease in HPCs, supporting the idea that a compensatory mechanism by Gpr97 is taking place when Gpr56 is absent. Preliminary in vivo data using a Gpr56 conditional KO mouse model corroborates our hypothesis. The data demonstrate that the Gpr97 performs a redundant function upon deletion of Gpr56 in the HS/PC population and the deletion of both Gpr56 and Gpr97 is sufficient to impair the production of HS/PC and HC in vitro. We conclude that GPR signalling through Gpr56 and/or Gpr97 is required for HPC production.

Study of TIM-3 in Pathogenesis of Myelodysplastic Syndrome

Objective The biological characteristics of TIM3+ stem cells were further explored from the aspects of cell morphology, cytogenetics, and animal models, providing a theoretical basis for the early identification of malignant MDS clones.Methods The subjects of the study were 44 newly diagnosed MDS patients who were admitted to the General Hospital of Tianjin Medical University from July 2016 to February 2018 and 17 normal controls. TIM3+ and TIM3-stem cells were sorted from the bone marrow of 10 unteated patients with MDS, CD34+ stem cells were sorted out from the bone marrow of four normal controls. We subjected them to Wright's staining, colony-forming unit culture, and fluorescence in situ hybridization (FISH) respectively. The TIM3+ , TIM3- stem cells, and MDSC in the bone marrow of 8 unteated patients with MDS were sorted. TIM-3 inhibitors, Gal-9 inhibitors, and both of the two inhibitors were added into the three kinds of cells separately and they were cultured in groups. We examined the apoptosis of TIM3+ stem cells and TIM3- stem cells, and then analyzed the role of TIM-3/Gal-9 pathway in TIM3+ stem cells and MDSC in patients with MDS. We sorted TIM3+ , TIM3-stem cells, and MDSC from the bone marrow of 11 untreated patients with MDS and sorted CD34+ stem cells from the bone marrow of 5 normal controls. The above cells were transplanted into 32 NCG mice. 10-12 weeks later, the differentiation of human-derived cells was detected by flow cytometry.Results The TIM3+ and TIM3- stem cells were stained by Wright's stain respectively and observed under high magnification are same. The CFU assay showed that the average number of BFU-E formed by TIM3+ stem cells, TIM3- stem cells and normal bone marrow stem cells was (6 vs 11 vs 12), and the CFU-E was (3 vs 6 vs 9), CFU-GM was (5 vs 17 vs 23). The expression of Gal-9 on MDSC was significantly higher than that of normal controls (0.62±0.64% vs 0.22±0.18%, P<0.05). The proportion of apoptotic TIM3+ stem cells in the TIM3+ stem cells mixed MDSC group was significantly lower than that in the two inhibitor groups and that of apoptotic TIM3-stem cells in TIM3- mixed MDSC group [12.59(5.12, 31.24) vs 42.54(29.41, 58.39), 65.86(24.44, 99.43), P<0.05]. In the animal transplant model of our study, the expression rate of hCD45 in TIM3+ stem cells mixed MDSC group was significantly higher than that in TIM3+ , TIM3- stem cells group and normal control CD34+ stem cells group (4.99±5.47% vs 0.65±0.22%, 1.68±2.38%, 0.55±0.40%, P<0.05). The expression rate of hCD19 in TIM3+ stem cells group was significantly lower than that in TIM3-stem cells group and normal control CD34+ stem cells group (8.68±9.34% vs 27.46±18.06%, 37.38±16.14%, P<0.01); The expression rate of hCD19 in TIM3+ stem cells mixed MDSC group was significantly lower than that in TIM3-stem cells group and normal control CD34+ stem cells group (4.76±4.78% vs 27.46±18.06%, 37.38±16.14%, P<0.05). The expression rate of hCD33 in TIM3+ stem cells group was significantly lower than that in TIM3- stem cells group (11.34±14.70 % vs 22.45±8.73%, P<0.05). The expression of hCD33 in TIM3+ stem cells mixed MDSC group was significantly lower than that in TIM3-stem cells group (5.07±5.29% vs 22.45±8.73%, P<0.05).Conclusions(1) The TIM3+ and TIM3- stem cells of MDS patients are difficult to distinguish morphologically, but the colony forming ability of the two are obviously different, and the karyotype abnormalities are also different. Therefore the TIM3+ stem cells may be the earlier karyotype anomaly “malignant” stem cells.(2) The expression rate of Gal-9 on MDSC cells in bone marrow of MDS patients was significantly increased. The apoptotic rate of TIM3+ stem cells in TIM3+ stem cells and MDSC mixed culture group was significantly lower than that of TIM3- stem cells in TIM3-stem cells and MDSC mixed culture group. Blocking the TIM-3/Gal-9 pathway can inhibit the anti-apoptotic effect of MDSC on TIM3+ stem cells, providing a new target for the clearance of malignant clones in patients with MDS.(3) The number of human-derived cells detected was the largest. And MDSC can promote the expansion of TIM3+ stem cells. Human-derived cells in the TIM3+ stem cell group and the TIM3+ stem cell and MDSC mixed group cannot undergo normal myeloid and lymphoid differentiation as well as the TIM3- stem cell group or the normal control CD34+ stem cell group. It is speculated that TIM3+ stem cells may have clonal growth, while MDSC has a promoting effect. DisclosuresNo relevant conflicts of interest to declare.

Inhibition of endothelial progenitor cells may explain the high cardiovascular event rate in patients with rheumatoid arthritis.

BACKGROUND Rheumatoid arthritis (RA) patients may suffer cardiovascular (CV) events much more than the general population, and CV disease is the leading cause of death in patients with RA. Our hypothesis was that impaired function of endothelial progenitor cells may contribute to endothelial dysfunction and the clinical CV events of patients with RA. METHODS About 27 RA patients (9 males and 18 females) with an active disease and 13 healthy subjects who served as the control group (nine males and four females) were enrolled to this prospective study. The ability to grow in culture colony-forming units of endothelial progenitor cells (CFU-EPCs) was measured, as well as their endothelial function using high-resolution ultrasonography of the brachial artery, and levels of C reactive protein (CRP) in the serum. For statistical analysis, we used the Student's t-test. RESULTS As a group, patients with RA were older (P < 0.0001), had severe endothelial dysfunction (P<0.0001), with impaired ability to grow CFU-EPCs (P<0.0001), and a higher inflammatory state (P = 0001). No difference was observed in BMI. All RA patients had an active disease (DAS28 3.9 ± 0.9) for 9.2 ± 6.5 years. The same differences were observed in both genders. CONCLUSIONS Patients with RA had an impaired ability to grow EPCs and severe endothelial dysfunction. Inability to grow colonies of EPCs reflects the impaired regenerative capacity of patients with RA and may explain the endothelial dysfunction and the high CV event rate among patients with RA.

Inhibition of endothelial progenitor cells may explain the high cardiovascular event rate in patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) patients may suffer cardiovascular (CV) events much more than the general population, and CV disease is the leading cause of death in patients with RA. Our hypothesis was that impaired function of endothelial progenitor cells may contribute to endothelial dysfunction and the clinical CV events of patients with RA. About 27 RA patients (9 males and 18 females) with an active disease and 13 healthy subjects who served as the control group (nine males and four females) were enrolled to this prospective study. The ability to grow in culture colony-forming units of endothelial progenitor cells (CFU-EPCs) was measured, as well as their endothelial function using high-resolution ultrasonography of the brachial artery, and levels of C reactive protein (CRP) in the serum. For statistical analysis, we used the Student's t-test. As a group, patients with RA were older (P < 0.0001), had severe endothelial dysfunction (P<0.0001), with impaired ability to grow CFU-EPCs (P<0.0001), and a higher inflammatory state (P = 0001). No difference was observed in BMI. All RA patients had an active disease (DAS28 3.9 ± 0.9) for 9.2 ± 6.5 years. The same differences were observed in both genders. Patients with RA had an impaired ability to grow EPCs and severe endothelial dysfunction. Inability to grow colonies of EPCs reflects the impaired regenerative capacity of patients with RA and may explain the endothelial dysfunction and the high CV event rate among patients with RA.

Lack of functional selectin-ligand interactions enhances innate immune resistance to systemic Listeria monocytogenes infection.

Selectin-ligand interactions are important for leukocyte homing and functionality. The roles of selectin-ligand interactions in modulating immunity to intracellular infections are not completely understood. Mice lacking the expression of fucosyltransferase-IV and -VII (Fucosyltransferase-IV and -VII double knockout, FtDKO) exhibit deficient functionality of selectin-ligand interactions. We addressed the kinetics of infection and immunity to Listeria monocytogenes (LM), an intracellular pathogen, in FtDKO mice. These mice exhibited enhanced ability to clear infection and increased survival to a lethal dose of LM infection relative to wild-type (WT) C57BL/6J controls. This was associated with increased levels of neutrophils, monocytes, and dendritic cells (DCs) in the blood and/or infected organs. Adoptive transfer of bone marrow (BM) cells from FtDKO mice to WT mice resulted in enhanced neutrophil numbers and improved clearance of LM bacteria in recipients. In vivo depletion of myeloid innate immune cells, particularly neutrophils, monocytes, macrophages, and DCs, using anti-Ly-6G (RB6-8C5) monoclonal antibody, reduced the ability of FtDKO mice to curtail LM infection. Nevertheless, depletion using anti-Ly-6G (1A8) known to exclusively deplete neutrophils did not abrogate increased resistance of FtDKO mice to LM infection, suggesting a role for other myeloid innate immune cells in this model. Examination of BM hematopoietic progenitors through flow cytometry and cell culture colony-forming unit assay showed increased frequencies of granulocyte-macrophage progenitors in FtDKO relative to WT mice, Overall, our results indicate that functional selectin ligand deficiency enhances innate immune-mediated resistance to systemic LM infection despite defective leukocyte migration and lymphocyte homing.

Pilot studies of a human BCG challenge model

Despite the great effort to develop an effective vaccine against tuberculosis (TB) there is currently no reliable and safe human challenge model that can be used for in vivo evaluation of new TB vaccine candidates and/or elucidation of the mechanisms of TB protective immunity. In this study, five volunteers were challenged with BCG intradermally (ID). Swab specimens were collected at multiple time points from the vaccination site pre- and post-vaccination to quantitate mycobacterial shedding as a surrogate of in vivo mycobacterial immunity. We compared the performance of the TaqMan qPCR assay against colony-forming unit cultures on 7H10 agar plates, and time to positivity (TTP) of mycobacterial growth indicator tubes (MGIT) in order to evaluate the reproducibility and sensitivity in measuring BCG burden in swab specimens. BCG was detected in swab specimens from all five volunteers by at least one method, and no single method was superior in terms of sensitivity and reproducibility. A comparison of all three methods showed significant correlations by Spearman's rank test between 7H10 agar plating and qPCR (R = 0.601, P = 0.00072), MGIT culture TTP and 7H10 agar plating (R = 0.412, P = 0.029) as well as MGIT culture TTP and qPCR (R = −0.708, P = 0.00003). However, the three methods were somewhat different with regard to early versus late detection of BCG shedding post-challenge. This ID BCG challenge model has unique potential to further explore correlations between reactogenicity and immune mechanisms involved in protection against mycobacterial infections, and could therefore become a reliable tool in the evaluation process of new TB vaccination strategies.

Loss of Adrenergic Nerves in the Bone Marrow Microenvironment Drives an Aging HSC Niche Phenotype

Hematopoietic stem cell (HSC) aging is accompanied by an expansion of myeloid-biased HSCs with declined long-term self-renewal functions, contributing to the development of hematologic malignancies. Recent studies have suggested a role for the bone marrow microenvironment in HSC aging but the extrinsic molecular mechanisms driving their aging remain largely unknown. Mesenchymal stem cells (MSCs) form a critical component of the HSC niche, promoting HSC quiescence and balanced differentiation. Some MSC activity is found associated with arterioles, which are densely innervated by the sympathetic nervous system (SNS). We used tridimensional whole-mount bone marrow confocal immunofluorescence imaging of HSCs and vascular structures to investigate the effects of aging on the HSC microenvironment. Comparative bone marrow analyses of aged (20-24 months) C57BL/6 and Nestin-GFP transgenic mice (where GFP marks MSCs) with young (2 month-old) C57BL/6 mice, revealed significant reductions in sympathetic innervation (3-fold, p<0.01) as determined by staining for tyrosine hydroxylase expressing nerve fibers. The SNS neuropathy of aged mice was accompanied with vascular remodeling characterized by an increase in total vascular density marked by CD31+/CD144+ (2-fold, p<0.01) and shortening of arterioles marked by CD31+/CD144+ and Nestin-GFP+ (5-fold reduction, p<0.001), reduction in α-smooth muscle actin (αSMA) pericytes (12-fold, p<0.01) and expansion of Nestin+ MSCs (4 fold, p<0.05). Whereas a subset of HSCs (~35%) are associated with arterioles in young mice, analyses of HSC localization in aged bone marrow revealed that HSCs were redistributed and expanded away from arterioles (p=0.0047). Functional analysis revealed a diminished in vitro clonal capacity of aged MSCs (determined by CFU-F and mesensphere assays; 3-fold, p<0.0001), reduced HSC maintenance activity determined by the ability to produce HSC maintenance factors (Cxcl12, Scf and Angpt1, 2-fold, p<0.01), and increased proliferative state (2-fold, p<0.0001), all indicative of reduced activity. Since SNS nerves regulate circadian egress of HSCs through regulation of MSC activity, we evaluated the circadian oscillations of HSCs in blood and niche factors in Nestin-GFP+ stromal cells. Aged mice exhibited ablated oscillations of progenitor release determined by peripheral blood colony-forming units in culture (CFU-C/ml blood) and phenotypic progenitors (LSK/ml blood). Furthermore, unlike the robust circadian oscillations of Cxcl12 levels of young MSCs, no circadian expression pattern was detected in aged mice, suggesting that the SNS neuropathy associated with aging has functional consequences by leading to disrupted circadian rhythms and expanded MSC populations with reduced HSC maintenance activity. To determine whether the loss of SNS nerves drives bone marrow vascular remodeling and HSC aging, we surgically sympathectomized young Nestin-GFP mice by unilateral microsection of both the sciatic and femoral nerves, while the sham-operated contralateral side served as control. Strikingly, four months following surgery, phenotypic HSCs selectively expanded in sympathectomized femurs (2-fold, p<0.05) and exhibited myeloid-biased differentiation upon competitive bone marrow transplantation compared to control (3-fold, p<0.01). Furthermore, similar to our observations in aged mice, chronic surgical sympathectomy (16 weeks) induced dramatic "aging-like" alterations of bone marrow arteriolar structures characterized with arteriolar shortening and loss of αSMA+ and Nestin-GFP+ arterioles (4-fold, p<0.01; 3 fold, p<0.001). These results thus suggest that aging-associated sympathetic neuropathy and the loss of β-adrenergic signals in the bone marrow microenvironment drive aging of HSCs by remodeling vascular niches and thus represent a potent therapeutic target for stem cell rejuvenation. DisclosuresFrenette:Pfizer: Consultancy; GSK: Research Funding; PHD Biosciences: Research Funding.

Enforced Expression of HOXB4 in Human Embryonic Stem Cells Enhances the Production of Hematopoietic Progenitors but Has No Effect on the Maturation of Red Blood Cells.

: We have developed a robust, Good Manufacturing Practice-compatible differentiation protocol capable of producing scalable quantities of red blood cells (RBCs) from human pluripotent stem cells (hPSCs). However, translation of this protocol to the clinic has been compromised because the RBCs produced are not fully mature; thus, they express embryonic and fetal, rather than adult globins, and they do not enucleate efficiently. Based on previous studies, we predicted that activation of exogenous HOXB4 would increase the production of hematopoietic progenitor cells (HPCs) from hPSCs and hypothesized that it might also promote the production of more mature, definitive RBCs. Using a tamoxifen-inducible HOXB4-ER(T2) expression system, we first demonstrated that activation of HOXB4 does increase the production of HPCs from hPSCs as determined by colony-forming unit culture activity and the presence of CD43(+)CD34(+) progenitors. Activation of HOXB4 caused a modest, but significant, increase in the proportion of immature CD235a(+)/CD71(+) erythroid cells. However, this did not result in a significant increase in more mature CD235a(+)/CD71(-) cells. RBCs produced in the presence of enhanced HOXB4 activity expressed embryonic (ε) and fetal (γ) but not adult (β) globins, and the proportion of enucleated cells was comparable to that of the control cultures. We conclude that programming with the transcription factor HOXB4 increases the production of hematopoietic progenitors and immature erythroid cells but does not resolve the inherent challenges associated with the production of mature adult-like enucleated RBCs. As worldwide blood donations decrease and transfusable transmitted infections increase, intense interest has ensued in deriving red blood cells (RBCs) in vitro from alternative sources such as pluripotent stem cells. A translatable protocol was developed to generate RBCs; however, these RBCs have an immature phenotype. It was hypothesized that the transcription factor HOXB4 could enhance their production and maturation. Although HOXB4 increased the production of erythroid progenitors, it did not promote their maturation. Despite the remaining challenges, a robust system has been established to test other candidates and add to the knowledge base in this field.

Mobilisation and dysfunction of haematopoietic stem/progenitor cells after Listonella anguillarum infection in ayu, Plecoglossus altivelis

Haematopoietic stem/progenitor cells (HSPCs) can mobilise into blood and produce immune cell lineages following stress. However, the homeostasis and function of HSPCs after infection in teleosts are less well known. Here, we report that Listonella anguillarum infection enhances HSPC mobilisation and reduces their differentiation into myeloid cells in ayu (Plecoglossus altivelis), an aquacultured teleost in East Asia. We established a colony-forming unit culture (CFU-C) assay to measure HSPCs using conditioned medium from peripheral blood mononuclear cells stimulated with phytohaemagglutinin. The number of CFU-Cs decreased in the head kidney and increased in the blood and spleen of ayu infected with L. anguillarum. HSPC mobilisation after L. anguillarum infection was mediated by norepinephrine. Furthermore, HSPCs from ayu treated with L. anguillarum lipopolysaccharide (LPS) showed defective myeloid differentiation and could no longer rescue L. anguillarum-infected ayu. HSPC expansion was suppressed after L. anguillarum infection or its LPS treatment in vitro. These results reveal a link between HSPC regulation and pathogen infection in teleosts.

Differentiation of haemopoietic stem cells and progenitor cells from human pluripotent stem cells

BackgroundInduced pluripotent stem cells hold great promise as an unlimited source of clinical grade immunologically matched haemopoietic stem cells (HSCs) for transplantation. However, there are currently no robust, reproducible differentiation protocols for the production of engraftable HSCs. We aimed to characterise the production of haemopoietic progenitor cells (HPCs) and HSCs during differentiation of human embryonic stem cells and induced pluripotent stem cells. MethodsWe used a serum-free, feeder-free, good manufacturing practice-compliant differentiation protocol established by the Novosang Consortium for the production of red blood cells for transfusion. Cell phenotype was assessed by flow cytometry for markers of HPCs and HSCs. Cell function was assessed in vitro by the colony forming units in culture (CFU-C) assay and in vivo with a long-term transplantation assay. FindingsAfter preliminary studies, the work focused on days 6, 8, and 10 of the differentiation protocol. In seven independent experiments, HPCs were reproducibly produced at high levels with over 90% of cells expressing CD43 by day 10. Almost all CD41+ cells coexpressed CD43 at all timepoints (day 10, 65% CD43+CD41+). CFU-Cs emerged in large numbers from day 6 and increased by day 10 (mean 1000 colonies per 105 cells plated [SD 478], six experiments). Cytospin and flow cytometry confirmed production of erythroid, granulocyte and macrophage, macrophage, and mixed colonies. The human embryonic HSC markers—vascular endothelial cadherin and CD45—were coexpressed at low levels (mean 3·6% [SD 2·5], seven experiments). No human engraftment was detected when cells were injected into immune-deficient NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice. InterpretationThis differentiation protocol reproducibly gave a high yield of HPCs. However, there was no evidence of engraftable HSCs being produced. This finding could indicate inadequate extrinsic signalling during differentiation. Further work will therefore focus on co-culturing human pluripotent stem cells with human stromal cells in an attempt to mimic the embryonic HSC niche. FundingWellcome Trust.

Mobilization of hematopoietic stem cells with highest self-renewal by G-CSF precedes clonogenic cell mobilization peak

Harvest of granulocyte colony-stimulating factor (G-CSF)-mobilized hematopoietic stem cells (HSCs) begins at day 5 of G-CSF administration, when most donors have achieved maximal mobilization. This is based on surrogate markers for HSC mobilization, such as CD34(+) cells and colony-forming activity in blood. However, CD34(+) cells or colony-forming units in culture (CFU-C) are heterogeneous cell populations with hugely divergent long-term repopulation potential on transplantation. HSC behavior is influenced by the vascular bed in the vicinity of which they reside. We hypothesized that G-CSF may mobilize sequentially cells proximal and more distal to bone marrow venous sinuses where HSCs enter the blood. We addressed this question with functional serial transplantation assays using blood and bone marrow after specific time points of G-CSF treatment in mice. We found that in mice, blood collected after only 48 hours of G-CSF administration was as enriched in serially reconstituting HSCs as blood collected at 5 days of G-CSF treatment. Similarly, mobilized Lin(-)CD34(+) cells were relatively enriched in more primitive Lin(-)CD34(+)CD38(-) cells at day 2 of G-CSF treatment compared with later points in half of human donors tested (n = 6). This suggests that in both humans and mice, hematopoietic progenitor and stem cells do not mobilize uniformly according to their maturation stage, with most potent HSCs mobilizing as early as day 2 of G-CSF.

Evidence of a Sophisticatedly Heterogeneous Population of Human Umbilical Vein Endothelial Cells

Induction and promotion of angiogenesis play a role in a diverse range of physiologic and pathophysiologic processes that are especially relevant to the field of regenerative medicine. For assessing vasculogenesis and neo-angiogenesis, identifying angiogenic factors, angiocrine factors, and vascular niche, facilitating tissue-repair and tumor growth, efficiently generating induced pluripotent stem cells, and coculturing with organ-specific stem cells, isolation and characterization of the subpopulation of human umbilical vein endothelial cells (HUVECs) and their endothelial progenitor cells (EPCs) are needed. In this study, primary HUVECs were collected from fresh umbilical cords and fractionated and characterized with the use of flow cytometry. Clonal colony assay showed that endothelial colony-forming units in culture frequently existed in fresh HUVECs. Antigenic profiling demonstrated that undifferentiated EPCs in HUVECs had normal endothelial marker CD31 with a subpopulation of cells positive for hematopoietic stem cell marker CD34 and c-Kit. With continuing passages, EPC markers CD34 and vascular endothelial growth factor receptor 2 expression decreased dramatically. Moreover, a distinct subpopulation with different proliferative capability and angiogenesis from the early-passage HUVECs was shown. In conclusion, it is possible to isolate accurately and to enrich EPCs or hematoangioblast-like cells from a heterogeneous population of HUVECs, and to explore the differential process with flow cytometry for further investigations.

Splenectomy increases the number of circulating hematopoietic stem/progenitor cells in patients with hepatitis C virus‐associated liver cirrhosis

The spleen is not believed to contribute to hematopoiesis in healthy adults. However, several reports have demonstrated that the spleen in adults contains a large number of hematopoietic stem/progenitor cells (HSC). Although splenectomy increases platelet and leukocyte counts, the effects of splenectomy on circulating HSC have not been elucidated. In this study, we evaluated the association between the number of circulating HSC and splenectomy in patients with hepatitis C virus (HCV)-associated liver cirrhosis (LC). In 48 patients with various stages of HCV-associated chronic liver disease and seven patients with LC who underwent splenectomy, and 10 healthy volunteers, we determined the numbers of circulating CD34+ cells and colony-forming unit culture by flow cytometry and methylcellulose culture, respectively. Plasma stromal cell-derived factor-1α (SDF-1α) concentrations were measured using an enzyme-linked immunosorbent assay. The numbers of circulating CD34+ cells and colony-forming unit culture decreased but the plasma SDF-1α concentration increased with the progression of liver disease. There was an inverse correlation between the number of circulating HSC and the plasma SDF-1α concentration. The numbers of circulating HSC and platelets were determined before and after splenectomy in seven patients with LC. In these patients, the numbers of circulating HSC and platelets increased significantly after splenectomy and the enhancing effect persisted for a long time. Our data suggest that the spleen plays an important role in modulating HSC dynamics in patients with HCV-associated chronic liver disease. Our results also imply that splenectomy may improve liver function in patients with LC.

In-vitro hematological toxicity prediction by colony-forming cell assays

Hematotoxicology is concerned with the adverse effects of xenobiotics on hematopoietic processes in a living system. In vitro tests for hematotoxicity have been applied in toxicity assessment of chemicals, drugs, food supplements and environment related studies. Hematopoietic Progenitor Colony-Forming Cell (CFC) assays and Stromal Cell Assays (also called as Non-hematopoietic Progenitor Assays) are already being used to detect hematological toxicities induced by different contaminants. These in vitro tests have been very useful in reducing the number of animals required for hematotoxicity testing. In this review, applications, limitations and future prospective of in vitro tests for hematotoxicity with emphasis on the techniques involved in the Colony forming unit culture systems are described.